Pharmacokinetics and brain uptake of a genetically engineered bifunctional fusion antibody targeting the mouse transferrin receptor.
Pharmacokinetics and brain uptake of a genetically engineered bifunctional fusion antibody targeting the mouse transferrin receptor.
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DOI：
10.1021/mp900235k
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发表时间：
2010-02-01
影响因子：
4.9
通讯作者：
Pardridge WM
中科院分区：
文献类型：
--
作者：
Boado RJ;Zhou QH;Lu JZ;Hui EK;Pardridge WM
单克隆抗体（MAb）是治疗脑部疾病的潜在新疗法。然而，MAb不能穿过血脑屏障（BBB）。本研究描述了一种由治疗性单链Fv （ScFv）抗体和小鼠/大鼠抗小鼠转铁蛋白受体（TfR）嵌合单抗组成的融合蛋白的基因工程。TfRMAb作为分子特洛伊木马，在小鼠体内通过血脑屏障运送治疗性ScFv。ScFv被融合到嵌合TfRMAb重链的羧基端，该融合蛋白被命名为cTfRMAb-ScFv。永久转染中国仓鼠卵巢细胞，在无血清培养基中克隆出高分泌细胞系。将cTfRMAb-ScFv融合蛋白纯化至凝胶均质，并用蛋白G亲和层析进行Western blotting。cTfRMAb-ScFv融合蛋白具有双重功能，通过ELISA和小鼠TfR测定，并通过放射受体测定，可以结合靶抗原。用Bolton-Hunter试剂对cTfRMAb-ScFv融合蛋白进行放射性碘化处理，小鼠药代动力学研究表明，融合蛋白在175±32分钟的中位停留时间内迅速从血液中清除。与无受体特异性的单抗相比，融合蛋白被脑吸收的百分比（ID）为3.5±0.7，而无受体特异性的单抗为0.06±0.01 %ID/g。这些研究表明，治疗性单抗可能被重新设计为血脑屏障分子特洛伊木马的融合蛋白，以便在体内靶向递送血脑屏障。
Monoclonal antibodies (MAb) are potential new therapeutics for brain diseases. However, MAb’s do not cross the blood-brain barrier (BBB). The present work describes the genetic engineering of a fusion protein comprised of a therapeutic single chain Fv (ScFv) antibody and a mouse/rat chimeric MAb against the mouse transferrin receptor (TfR). The TfRMAb acts as a molecular Trojan horse to ferry the therapeutic ScFv across the BBB in vivo in the mouse. The ScFv is fused to the carboxyl terminus of the heavy chain of the chimeric TfRMAb, and this fusion protein is designated cTfRMAb-ScFv. Chinese hamster ovary cells were permanently transfected, and a high secreting cell line in serum free medium was cloned. The cTfRMAb-ScFv fusion protein was purified to homogeneity on gels and Western blotting with protein G affinity chromatography. The cTfRMAb-ScFv fusion protein was bi-functional and bound both the target antigen, as determined by ELISA, and the mouse TfR, and as determined with a radio-receptor assay. The cTfRMAb-ScFv fusion protein was radio-iodinated with the Bolton-Hunter reagent, and a pharmacokinetics study in mice showed the fusion protein was rapidly cleared from blood with a median residence time of 175 ± 32 min. The fusion protein was avidly taken up by brain with a % injected dose (ID)/g of 3.5 ± 0.7, as compared to an MAb with no receptor specificity, which was 0.06 ± 0.01 %ID/g. These studies demonstrate that therapeutic MAb’s may be re-engineered as fusion proteins with BBB molecular Trojan horses for targeted delivery across the BBB in vivo.
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影响因子：
3.8
作者：
Boado, Ruben J.;Zhang, Yun;Wang, Yuntao;Pardridge, William M.
通讯作者：
Pardridge, William M.
DOI：
10.1073/pnas.151261398
发表时间：
2001-07-17
影响因子：
11.1
作者：
DeMattos, RB;Bales, KR;Holtzman, DM
通讯作者：
Holtzman, DM
影响因子：
3.8
作者：
Boado, Ruben J.;Zhang, Yufeng;Pardridge, William M.
通讯作者：
Pardridge, William M.
影响因子：
5.3
作者：
Lombardo, JA;Stern, EA;Hyman, BT
通讯作者：
Hyman, BT
影响因子：
2.9
作者：
SKARLATOS, S;YOSHIKAWA, T;PARDRIDGE, WM
通讯作者：
PARDRIDGE, WM